Wound and ulcer treatment with super-oxidized water

ABSTRACT

Super-oxidized water based on hypochlorous acid, such as is obtained by the electrochemical treatment of a saline solution, may be used in the treatment of leg ulcers or other open wounds. Preferably, the pH of the super-oxidized water is in a range of 4 to 7, and the water has a redox potential of &gt;950 mV. Medicaments based on the super-oxidized water may be in liquid or gel form. The super-oxidized water is able to control the microbial population within the wound and at the same time permit cell proliferation.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of co-pending U.S. Pat. Ser. No.10/084,518, filed Feb. 25, 2002, now abandoned, which is a continuationof International Patent Application No. PCT/GB00/03264, filed Aug. 23,2000, which was published in the English language on Mar. 1, 2001, underInternational Publication No. WO 01/13926 A2, and the disclosure ofwhich is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] This invention relates to mixtures of oxidants which are referredto in this specification as “super-oxidized water,” a term which isknown in the art.

[0003] Super-oxidized water may be used as a sterilizing, disinfectingand biocidal solution. One form of super-oxidized water is produced bythe applicant under the trademark STERILOX®. This STERILOXsuper-oxidized water is generated at the point of use, for example in ahospital, by passing saline solution over coated titanium electrodesseparated by a semi-permeable ceramic membrane at a current of about 6to 9 Amps. An apparatus having coated titanium electrodes separated by asemi-permeable ceramic membrane is disclosed in the specifications of UKPatent Nos. 2253860 and 2274113. The basic structure of the apparatus isdisclosed in GB2253860 and can be used to produce the STERILOXsuper-oxidized water.

[0004] STERILOX super-oxidized water contains a mixture of oxidizingspecies, predominantly hypochlorous acid (HOCI) and sodium hypochlorite.The STERILOX super-oxidized water has a pH of 5-7 and an oxidationreduction potential (redox) of around 1000 mV. The high redox potentialallows for the quick and efficient destruction of microbes (bacteria,viruses, fungi and spores). Hypochlorous acid and hypochlorite are inequilibrium and the position of the equilibrium is determined solely bythe pH.

[0005] Applicant has found that the resultant super-oxidized water isnon-hazardous, non-irritating and non-sensitizing to the skin,non-irritating to the eyes, not harmful if swallowed and shows noevidence of mutagenic activity.

[0006] It is considered that hypochlorous acid exerts its biocidaleffect by attacking the surface and plasma membrane proteins, impairingtransport of solutes and the salt balance of bacterial cells (Pietersonet al, Water SA, 22(1): 43-48 (1996)). However, it is believed that HOCldoes not enter freely into eukaryotic cells, which may explain theselectivity of hypochlorous solutions.

[0007] The STERILOX process produces an extremely effective sterilizing,cold, non-toxic solution, which is free from highly toxic chemicals andacts against a wide variety of bacteria, fungi, viruses and spores. Thegeneration of STERILOX solutions requires only water, electricity andpure, vacuum-dried crystalline salt. Applicant considers that theSTERILOX super-oxidized water will be suitable for a broad range ofapplications in both medical and non-medical environments, such as thepreservation of poultry and fish and general agricultural andpetrochemical uses, the breaking down of bacterial biofilm, watertreatment and general disinfection in medical and veterinaryapplications. The STERILOX super-oxidized water has been found to beparticularly useful for the disinfection of endoscopes which aresensitive to other cold disinfectants, such as peracetic acid, which arecommonly used.

[0008] While glutaraldehyde may be used as a reliable disinfecting agentof flexible fiber-optic endoscopes and other heat-sensitive instruments,although being widely practiced in many hospitals, its use can causeasthma and dermatitis in healthcare staff as a result of exposure toglutaraldehyde fumes, hence a predilection to the use of peracetic acidand the relatively recent move towards the use of STERILOXsuper-oxidized water in such applications.

[0009] STERTLOX super-oxidized water has been tested and is the subjectof two scientific papers by Selkon et al, Journal of Hospital Infection,41: 59-70 (1999) and Shetty et al, Journal of Hospital Infection, 41:101-105(1999). In these studies, freshly produced STERILOXsuper-oxidized water was found to be highly active against Mycobacteriumtuberculosis, Mycobacterium avium-intracellulare, Mycobacteriumchelonae, Escherichia coli (including type 0157), Enterococcus faecalis,Pseudomonas aeruginosa, Bacillus subtilis var niger spores,methicillin-resistant Staphylococcus aureus, Candida albicans,poliovirus type 2 and human immunodeficiency virus HIV-1.

[0010] There has been a recent upsurge in interest in the use ofsuper-oxidized water as a disinfectant, because of its rapid and highlybiocidal activity against a wide range of bacteria. Tanaka et al,Journal of Hospital Infection. 34: 43-49 (1996), report the electrolysisof a saline solution to produce a super-oxidized water with a highlyacidic pH of 2.3-2.7, which limits its suitability for manyapplications, particularly the disinfection of endoscopes. The acidic pHof the super-oxidized water produced by the method described by Tanakaet al. also precludes its use in other medical indications.

[0011] Having carried out trials in a large number of applications,including those mentioned above, Applicant turned its attention to theuse of STERILOX super-oxidized water as a disinfectant of mammaliantissue, in particular the treatment of open wounds such as leg ulcers.

[0012] An article by Cherry in The Prescriber (May 1996) entitled “GPguide to the care of patients with leg ulcers” states “leg ulcers arenotoriously difficult to treat successfully and can seriously reduce thepatient's quality of life.” Indeed, according to Cherry,“epidemiological studies have shown that at any given time there areapproximately 100,000 patients in the UK that have leg ulceration. Intreating these patients it has been estimated that over £39 million peryear alone is spent on materials used in their ulcer care.”

[0013] There are two types of leg ulcers: arterial and venous. Arterialulcers, which are much harder to treat, are caused by ischaemia, whilevenous ulcers are caused by blood stasis in the veins.

[0014] There are many proposals for the management of ulcers, all ofwhich have varying degrees of success. Successful ulcer management isvery much dependent on the rigid adherence to a program of treatment incombination with effective disinfection of the wound, which reducesbacterial infection and promotes the regeneration of dermal fibroblastsand keratinocytes in the bed of the ulcer which are essential forhealing of the wound and the growth of new tissue. If the bacterialgrowth is not controlled, the wound cannot heal.

[0015] The most useful treatment for venous ulcers is the use ofcompression bandages together with elevation of the leg(s). This mimicsthe pumping action of the calf muscles which return the blood back tothe body and maximizes the removal of blood from the leg(s). Inconjunction with this, other treatment strategies include the use oftopical treatments such as GRANUFLEX® to aid granulation and skinrepair, alginates to clean the wound of debris, dry inert dressings toprotect the wound (but which do not promote healing), and bacteriostaticor bactericidal ointments to reduce the infection. While antibioticshave been used to reduce infection in the past, nowadays this is not atreatment of choice due to the increased risk of antibiotic resistance.

[0016] While potassium permanganate (KMnO₄) is an oxidant which hasstood the test of time in the treatment of leg ulcers, it stillnevertheless has the disadvantages of irritating and injuring newlygrown skin and causing skin discoloration. Known hypochlorites, such asEUSOL (Edinburgh University Solution of Lime) and Daikin's solution,rely on a high concentration of hypochlorite ions for their disinfectantproperties. In fact, these compounds are no longer recommended for usedue to their irritant and painful effects and impairment of cell growthwhich outweigh their therapeutic value, resulting in these preparationsfalling out of use. Attempts have been made to reduce the alkalineeffect of the high hypochlorite ion content of these Solutions, e.g. bythe use of suitable buffers, but have been found to be ineffective insuch circumstances.

[0017] All this has militated against the use of preparations includinghypochlorites for the treatment of leg ulcers. However, the success indisinfection and sterilization of endoscopes and the known non-irritanteffects of the STERILOX super-oxidized water, have led the Applicant tore-address the treatment of open wounds such as leg ulcers.

BRIEF SUMMARY OF THE INVENTION

[0018] As a first step, in vitro tests were carried out on single layersof cultured human dermal fibroblast cells and keratinocyte cells toascertain whether or not super-oxidized water had any effect on theviability of the cells. The cells were incubated under sterileconditions in a super-oxidized water based on hypochlorous acid andincluding sodium hypochlorite and other oxidized chlorine species,having a pH range from 4 to 7 and a redox potential of around 1000 mV.

[0019] Surprisingly, Applicant found that there is an optimum pH atwhich cell growth is not inhibited despite there being other pH levelswithin the range at which the viability of the cells is impaired.Indeed, in view of the findings of Tanaka et al, Applicant expected thatthe lower pH range would be more effective.

[0020] The next step was crucial, because the applicant then had toascertain whether or not the in vitro results could not only bereplicated in vivo but also that there would be a sufficient biocidaleffect to counteract any bacterial activity which would prejudice theviability of new cells. Accordingly, a clinical trial was carried out ona patient with chronic venous leg ulcers using freshly producedsuper-oxidized water based on hypochlorous acid having a pH of 5.4.

[0021] The patient did not experience any pain, and in fact commentedthat the treatment was comfortable and had a soothing effect. There wasa positive effect on the bacterial flora as well as the clinicalappearance of the wounds. No adverse effect was observed on thesurrounding skin which, in a number of patients with long-standingulcers, is often sensitive.

[0022] Applicant believes that the effects which have been observed canbe explained by the low concentration of oxidized (free available)chlorine present in a super-oxidized water based on hypochlorous acid.This is in contrast to commonly known hypochlorite solutions which owetheir biocidal activity to a high concentration of free chlorine(including hypochlorite) as indicated by their characteristic smell.

[0023] It could be said that Applicant has discovered a principle, whichis that a balance between the biocidal effect and non-inhibition of cellgrowth enables the microbial population present in a wound to becontrolled such as to allow cell growth to occur.

[0024] In order to carry this principle into effect and from one aspectof the invention, there is provided a super-oxidized water which isbased on hypochlorous acid, acts as a biocide and allows cell growth.Expressed in another way, the present invention resides in asuper-oxidized water in which the biocidal effect is due to hypochlorousacid and the non-inhibitory effect on cell growth is dependent on thelevel of the pH of the hypochlorous acid solution.

[0025] From a still further aspect of the invention, in a super-oxidizedwater based on hypochlorous acid and having a pH of 4 to 7, the solutionhas a pH selected in a range of about 4.3 to 6.2.

[0026] In a further aspect of the invention, there is provided, for usein the treatment of, or medicament for, skin ulcers or other openwounds, a super-oxidized water or other formulation, such as a gel,which is based on hypochlorous acid, acts as a biocide and allows cellgrowth. The super-oxidized water or other preparation based onhypochlorous acid may have a pH of 4 to 7, with the pH preferably beingselected in a range of about 4.0 to 6.5, and more preferably in a rangeof about 4.0 to 6.2.

[0027] In any of the aspects of the invention defined above, thesuper-oxidized water has a biocide rate (D Value) of approximately 1 logunit reduction unit of bacillus subtilis spores in less than 1 minutewith a 9:1 super-oxidized water: innoculum mix. A biocide rate of about3.4 seconds may be achieved and is particularly preferred.

[0028] The super-oxidized water based on hypochlorous acid may beobtained by the electrolysis of a saline solution. Accordingly, fromanother aspect of the invention there is provided a method of obtaininga super-oxidized water based on hypochlorous acid, the method comprisingpassing the saline solution through an electrochemical cell havingelectrodes separated by a semi-permeable membrane and operating in suchmanner as to produce a super-oxidized water based on hypochlorous acidwhich acts as a biocide and allows cell growth.

[0029] In a preferred method of carrying out the invention, thesuper-oxidized water is obtained and the pH level of the solution isadjusted by a buffering action, which involves the use of an alkalinefeed from the cell. By using an alkaline solution in this way, theaddition of conventional buffers and the consequent expense is avoided.

[0030] While the pH of the super-oxidized water is preferably adjustedto be in a range of about 4.3 to 6.5, Applicant has found that, in thecase of wounds, such as leg ulcers, it is advantageous if the pH isadjusted to about 5.4.

[0031] From a still further aspect, the invention resides in a method ofproducing a medicament containing super-oxidized water as defined abovefor use in the treatment of leg ulcers or other open wounds.

[0032] The invention also comprehends, in a super-oxidized water orother formulation having a pH of 4 to 7, the selection of a pH in arange of about 4.3 to 6.5, and preferably about 5.4, that is used forthe treatment of leg ulcers or other open wounds.

[0033] By means of any of the aspects of the invention defined above,the disinfection of wounds such as leg ulcers is readily achievedwithout irritation and pain, the spread of infection is reduced, andcell growth and regeneration are facilitated, thereby enhancing healing.An added advantage is that there is no resistance or tolerance developedto disinfection which would occur with antibiotics.

[0034] Furthermore, the super-oxidized water of the invention lendsitself readily to the treatment being carried out in many ways, althoughit has been found that a hydrobath in which the leg is immersed issoothing and generally pleasant for the patient. However, it should beappreciated that preparations other than solutions may be used, forexample gels.

[0035] From yet another aspect, the invention also resides in a methodof treating a human or animal body having a leg ulcer or other openwound using any of the super-oxidized water based on hypochlorous acidherein referred to above and any of the methods defined hereinabove.

[0036] Applicant believes that the present invention, in any or all ofits aspects is a breakthrough and will revolutionize the treatment ofopen wounds, in particular leg ulcers, in a way which has been found tobe impossible hitherto.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0037] The foregoing summary, as well as the following detaileddescription of the invention, will be better understood when read inconjunction with the appended drawings. For the purpose of illustratingthe invention, there are shown in the drawings embodiments which arepresently preferred. It should be understood, however, that theinvention is not limited to the precise arrangements andinstrumentalities shown. In the drawings:

[0038]FIG. 1a is a bar graph illustrating the effect on dermalfibroblast proliferation, measured by absorption assay at 3 and 6 days,of various concentrations super-oxidized water; and

[0039]FIG. 1b is a bar graph similar to FIG. 1a, showing the results ofthe absorption assay at 6 days.

DETAILED DESCRIPTION OF THE INVENTION

[0040] In order that the invention may be more readily understood,reference will now be made, by way of example, to the accompanyingexamples, in which:

[0041] Example 1 describes an in vitro study, which investigated theeffect of super-oxidized water based on hypochlorous acid on theproliferation of cultured human dermal fibroblast cells.

[0042] Example 2 describes an in vitro study, which investigated thecytotoxic effect of super-oxidized water based on hypochlorous acid oncultured human dermal fibroblast cells.

[0043] Example 3 describes an in vitro study, which investigated theeffect of super-oxidized water based on hypochlorous acid on theproliferation of cultured human keratinocyte cells.

[0044] Example 4 describes a clinical trial of super-oxidized water on apatient with chronic leg ulcers.

EXAMPLE 1

[0045] Fibroblasts are flattened, irregular-shaped, connective tissuecells which are ubiquitous in fibrous connective tissue. They secretecomponents of the extracellular matrix, including collagen, and play animportant role in tissue regeneration.

[0046] Three in vitro trials of super-oxidized water based onhypochlorous acid were carried out on single-layer cell cultures ofhuman dermal fibroblast (HDF) cells to ascertain whether thesuper-oxidized water affected HDF cell proliferation. A range ofdilutions of super-oxidized water at different pH levels wasinvestigated.

[0047] Method

[0048] The super-oxidized water used in the trials was the product ofthe electrolysis of an aqueous saline solution passed over a mixture ofproprietary catalysts on titanium electrodes to give a mixture ofoxidizing species, particularly hypochlorous acid (HOCl) at aconcentration of about 144 mg/l to 400 mg/l available free chlorine(Cl). The super-oxidized water was produced as required for each test;the apparatus (supplied by Sterilox Medical Limited, Abingdon, UK) wasoperated to give a final solution redox potential of >950 mV asrecommended by the company. Appropriate dilutions of the super-oxidizedwater were made, and the pH of the final solution was adjusted using aphosphate buffer.

[0049] For the proliferation assay, HDF cells were seeded in normal(10%) fetal calf serum (FCS) and Dulbecco's Modified Eagle Medium (DMEM)at 1.5×10³ cells/well. After 24 hours the medium was changed to low(0.4%) FCS/DMEM. After a further 48 hours incubation super-oxidizedwater at varying concentrations was added to the cells. The viability ofthe cells was observed, using a standard absorption assay, 3 and 6 daysafter the application of super-oxidized water.

[0050] Results

[0051] i) Trial 1: HDF cells were incubated with super-oxidized water ina range of dilutions at apH of4.3. The dilutions used were: 1, ⅓,{fraction (1/7)}, {fraction (1/14)}, {fraction (1/28)}, {fraction(1/56)}, {fraction (1/112)}, {fraction (1/224)}, {fraction (1/448)},{fraction (1/896)}, {fraction (1/1792)} and 0.

[0052] As shown in the accompanying graphs, on both day 3 (FIG. 1a) andday 6 (FIG. 1b) super-oxidized water dilutions of {fraction (1/28)} orless significantly inhibited HDF proliferation or killed the cells.Slight inhibition of proliferation was seen at a dilution of {fraction(1/56)}. Dilutions of {fraction (1/112)} to {fraction (1/448)} showed noeffect on proliferation, while the {fraction (1/896)} dilution showedsome cell proliferation and the dilution of {fraction (1/1792)} showedsignificant proliferation of HDF cells.

[0053] These results show that high concentrations of super-oxidizedwater significantly inhibit HDF proliferation, probably because of theacidity, and therefore toxicity, of the super-oxidized water. The highlevel of proliferation seen with a concentration of super-oxidized waterat {fraction (1/1792)} may be attributed to other factors.

[0054] ii) Trial 2: Using the same conditions as Trial 1, HDF cells wereincubated with super-oxidized water in a range of dilutions at a pH of6.2. The dilutions used were: {fraction (1/20)}, {fraction (1/40)},{fraction (1/60)}, {fraction (1/80)}, {fraction (1/100)}, {fraction(1/120)}, {fraction (1/1000)}, {fraction (1/1500)}, {fraction (1/2000)},{fraction (1/3000)}, {fraction (1/4000)} and 0.

[0055] No stimulation of proliferation was seen and indeed, inhibitionof HDF growth was seen with cells incubated with a dilution ofsuper-oxidized water of {fraction (1/20)}. However, the higher dilutionof {fraction (1/40)} showed no cell toxicity.

[0056] This trial was repeated with the HDF cells seeded insuper-oxidized water at dilutions of 1, ¼, ⅛, {fraction (1/12)},{fraction (1/16)}, {fraction (1/20)}, {fraction (1/24)}, {fraction(1/28)} and {fraction (1/32)}. After both day 3 and day 6, cell damageor inhibition of proliferation was seen at dilutions of {fraction(1/20)} and below. However, dilution of more than {fraction (1/20)}showed no damage or inhibition or proliferation.

[0057] In conclusion, while the more alkaline pH appears to be lesstoxic to HDF cells, proliferation of HDF cells is not seen at this pH.

[0058] iii) Trial 3: In this trial, two plates of cells were grownalways in normal growth medium (10% FCS/DMEM). One plate of cells wastreated as described in Trial 1 but after three days of incubation withthe super-oxidized water, the growth medium was changed from 0.4%FCS/DMEM to 10% FCS/DMEM in order to observe the recovery of the cells.

[0059] HDF cells were incubated at 31° C. with super-oxidized water in arange of dilutions at a pH of 5.4. The dilutions used were: {fraction(1/10)}, {fraction (1/20)}, {fraction (1/40)}, {fraction (1/60)},{fraction (1/80)}, {fraction (1/100)}, {fraction (1/150)}, {fraction(1/1000)}, {fraction (1/1500)}, {fraction (1/2000)}, {fraction (1/4000)}and 0.

[0060] On day 3 and day 6 cell proliferation was seen in cells incubatedwith super-oxidized water at a dilution of {fraction (1/20)} or higherin either 0.4% or 10% FCS/DMEM. Some levels of proliferation had reachedstatistical significance. A dilution of {fraction (1/10)} super-oxidizedwater inhibited cell growth in HDF cells grown in 0.4% FCS/DMEM but notin 10% FCS/DMEM.

[0061] After 3 days incubation in 0.4% FCS/DMEM with or withoutsuper-oxidized water, the medium was changed to 10% FCS/DMEM. The cellsthat had been incubated with super-oxidized water showed the sameability to recover from depression of cell growth seen while growing in0.4% FCS/DMEM as control cells.

[0062] This trial was repeated with the HDF cells seeded insuper-oxidized water at dilutions of {fraction (1/7)}, {fraction(1/10)}, {fraction (1/15)}, {fraction (1/20)}, {fraction (1/40)},{fraction (1/60)}, {fraction (1/100)}, {fraction (1/500)}, {fraction(1/1000)}, {fraction (1/2000)}, {fraction (1/4000)} and 0. 100621 Again,on both day 3 and day 6, cell proliferation was seen with HDF cellsgrown in 0.4% FCS/DMEM with the difference seen being statisticallysignificant at most dilutions. No inhibition of cell growth was seen,even at the dilution of {fraction (1/7)} super-oxidized water.Stimulation of cell growth was also seen in cells grown in 10% FCS/DMEMin the presence of super-oxidized water. However, the levels ofproliferation did not reach statistical significance. Where cell growthhad been impaired by incubation with super-oxidized water, recovery wasseen, confirming the observations from the first set of experiments.

[0063] In summary, HDF cells incubated with super-oxidized water at pH5.4 showed no inhibition of cell growth, even in the presence of a{fraction (1/7)} dilution of super-oxidized water.

[0064] Conclusion

[0065] The presence of super-oxidized water at a pH of 5.4 does notinhibit HDF cell growth in vitro.

EXAMPLE 2

[0066] Three in vitro trials were carried out to investigate thecytotoxic effect of super-oxidized water based on hypochlorous acid onHDF cells.

[0067] Method

[0068] The super-oxidized water based on hypochlorous acid was identicalto that described in Example 1.

[0069] HDF cells were seeded in 10% FCS/DMEM at 5×10³ cells/well. Afterincubation at 31° C. for 72 hours, dilutions of super-oxidized waterwere prepared in HBSS and added to the cells. The viability of the cellswas ascertained by a standard absorption assay at time intervals of 15minutes up to one hour from the addition of the super-oxidized water.

[0070] Results

[0071] i) Trial 1: HDF cells were incubated with super-oxidized water ina range of dilutions at a pH of 4.3. The dilutions used were: 1, ⅓,{fraction (1/7)}, {fraction (1/14)}, {fraction (1/28)}, {fraction(1/56)}, {fraction (1/112)}, {fraction (1/224)}, {fraction (1/448)},{fraction (1/896)}, {fraction (1/1792)} and 0.

[0072] No effect on cell viability was seen in the presence ofsuper-oxidized water at dilutions of {fraction (1/28)} or more at any ofthe time points. A dilution of {fraction (1/14)} induced mild damage tothe cells while dilutions of {fraction (1/7)} and less killed the cells.

[0073] ii) Trial 2: Using the same conditions as trial 1, HDF cells wereincubated with super-oxidized water in a range of dilutions at a pH of6.2. The dilutions used were: 1, ¼, ⅛, {fraction (1/12)}, {fraction(1/16)}, {fraction (1/20)}, {fraction (1/24)}, {fraction (1/28)},{fraction (1/32)}, {fraction (1/36)}, {fraction (1/40)} and 0.

[0074] No significant effect was seen on the viability of HDF cells inthe presence of super-oxidized water at dilutions of {fraction (1/20)}or more at any time point. However, dilution of {fraction (1/16)} orless induced cell damage.

[0075] iii) Trial 3: Using the same conditions as Trials 1 and 2, HDFcells were incubated with super-oxidized water in a range of dilutionsat a pH of 4.0. The dilutions used were: 1, ¼, ⅛, {fraction (1/12)},{fraction (1/16)}, {fraction (1/20)}, {fraction (1/24)}, {fraction(1/28)}, {fraction (1/50)}, {fraction (1/100)}, {fraction (1/200)} and0.

[0076] Dilutions of super-oxidized water at {fraction (1/24)} and{fraction (1/20)} induced slight damage to the cells while dilution of{fraction (1/16)} or less induced cell death.

[0077] Conclusion

[0078] The results of these trials support the results shown in Example1 in that, while super-oxidized water at pH 4.0 to 4.3 and pH 6.2 inducedamage to cultured HDF cells, greater cytotoxic effects are seen at thelower pH.

EXAMPLE 3

[0079] In view of the results described in Examples 1 and 2, two invitro trials were carried out to investigate the effect super-oxidizedwater on human keratinocyte (HK) cell proliferation. The super-oxidizedwater used in these trials was identical to that described in Example 1.Keratinocytes are epidermal skin cells that synthesize keratin and,together with dermal fibroblasts, are essential for skin healing.

[0080] Trial 1

[0081] HK cells (subcultured, P2, FS, 7 years) were seeded at 8×10³cells/well and incubated at 31° C. in CLONETICS® (Biowhittaker, US)serum-free medium with complete supplements, hereinafter referred to askeratinocyte growth medium (KGM), in four 24-well plates. After 24 hoursincubation the medium in plates 1 and 2 was changed to CLONETICS®(Biowhittaker, US) serum-free medium without complete supplements,hereinafter referred to as keratinocyte basal medium (KBM).

[0082] After a further 48 hours incubation super-oxidized water dilutedin KBM at pH 5.4 was added to plates 1 and 2, and super-oxidized waterdiluted in KGM was added to plates 3 and 4. The dilutions ofsuper-oxidized water were: {fraction (1/10)}, {fraction (1/20)},{fraction (1/50)}, {fraction (1/100)}, {fraction (1/150)}, and 0. The pHof the final super-oxidized water solution was adjusted using aphosphate buffer.

[0083] After incubation for a further 3 days a standard absorption assaywas carried out on plate 3 to observe the viability and growth of thecells. The absorption assay was carried out on plate 4 after a stillfurther two days. Since the cells incubated in KBM did not grow well,plates 1 and 2 were discarded.

[0084] The absorption assay showed that, on both day 3 and day 5, cellproliferation had occurred in the presence of all dilutions ofsuper-oxidized water. At day 5, the level of cell proliferation hadreached a significant level compared to cell growth in the absence ofsuper-oxidized water.

[0085] Trial 2

[0086] In view of the fact that the HK cells did not grow in KBM andshowed significant proliferation in the presence of super-oxidized waterin KGM, it was decided to use KBM with lower concentrations ofsupplements as a holding medium, with or without super-oxidized water.

[0087] HK cells (thawed, P2, FS, 7 years) were seeded to six 96-wellplates at 3×10³ cells/well in KGM. After incubation for 24 hours, themedium in plates 1 and 2 was changed to KBM with 20% supplements, andthe medium in plates 3 and 4 was changed to KBM with 50% supplements.

[0088] After incubation for a further 24 hours, super-oxidized waterdiluted in KBM with 20% supplements was added to plates 1 and 2,super-oxidized water diluted in KBM with 50% supplements was added toplates 3 and 4, and plates 5 and 6 received super-oxidized water dilutedin complete KGM. The dilutions of super-oxidized water were: {fraction(1/7)}, {fraction (1/10)}, {fraction (1/15)}, {fraction (1/20)},{fraction (1/40)}, {fraction (1/60)}, {fraction (1/100)}, {fraction(1/500)}, {fraction (1/1000)}, {fraction (1/2000)}, {fraction (1/4000)}and 0.

[0089] The cells were incubated for a further 3 days in the presence ofsuper-oxidized water, after which time, a standard absorption assay wascarried out on plates 1, 3 and 5 to ascertain the viability of thecells, and the medium in plates 2, 4 and 6 was changed to KGM. Plates 2,4 and 6 were assayed after a further 48 hours of incubation.

[0090] Stimulation of cell proliferation on both day 3 and day 5 wasseen in all percentages of KGM supplements. However, the level ofstimulation was not significantly different when compared to controlcell growth. No cytotoxicity was seen even at the low dilution of{fraction (1/7)} super-oxidized water.

[0091] Conclusion

[0092] Dermal keratinocytes cultured in the presence of KGM andsuper-oxidized water showed enhanced cell proliferation, and nocytotoxicity was seen in the presence of super-oxidized water.

EXAMPLE 4

[0093] A preliminary clinical evaluation of super-oxidized water basedon hypochlorous acid was carried out on one patient with chronic venousulcers on both left and right legs. The aim of the trial was todetermine whether the bacterial status of the ulcers is altered and thebed of the ulcer improved by treatment with super-oxidized water.

[0094] Method

[0095] The patient's legs were immersed in 40 liters of super-oxidizedwater in a hydrobath for fifteen minutes before being allowed to dry. Anintermediate assessment without treatment was carried out after oneweek.

[0096] A second treatment with super-oxidized water was repeated aftertwo weeks in which the patient was subjected to three fifteen-minutewashes at approximately three-hour intervals. Post-treatment clinicalevaluation was carried out one and several days after the secondtreatment.

[0097] Semi-quantitative microbiological analysis of the leg ulcers wascarried out on swabs taken before and fifteen minutes after treatmentwith super-oxidized water.

[0098] Results

[0099] After the first treatment, the patient reported that thetreatment was comfortable and free from pain. The appearances of theulcers on both legs were markedly improved when assessed five hoursafter treatment.

[0100] As shown in Table 1, quantitative microbiology showed a reductionin the number of colony-forming units in the order of 102 in the rightleg ulceration and a reduction in the order of 104 on the left leg.TABLE 1 semi-quantitative microbiological analysis of leg ulcers beforeand after treatment with super-oxidized water based on hypochlorousacid. Figures quoted indicate the number of colony-forming units (cfu)per ml. Right Leg Left Leg Pre-treatment 1 × 10⁷ 1 × 10⁷ Post-treatment1 × 10⁵ 1 × 10³

[0101] The patient was seen one week after the first treatment and wastreated with conventional therapy, including potassium permanganate. Theeffect of these on the appearance of the leg ulcers following treatmentdid not appear to be as striking as that seen with super-oxidized water.

[0102] A second treatment with super-oxidized water was repeated afurther week later, and similar beneficial results were obtained. Inbetween the treatment periods the ulcers had become sloughy on bothlegs. Immediately after the first wash, the ulcer bed was whitish due toeffervescence. A cleansing effect was seen after the later two washes,and a marked improvement was seen with the state of the ulcer 18 hoursafter the first wash.

[0103] The patient reported no discomfort to the treatment, the solutionin the bath was soothing, and the skin felt a bit tight afterwards. Thepatient commented that the tightness started to be felt once cold airwas accessible to the skin.

[0104] Referring to Table 2, quantitative microbiology showed areduction in the number of colony-forming units in the order of 102 inthe right leg ulceration and a reduction in the order of 104 on the leftleg. TABLE 2 semi-quantitative microbiological analysis of leg ulcersbefore and after treatment with super-oxidized water based onhypochlorous acid. Figures quoted indicate the number of colony-formingunits (cfu) per ml. Right leg organisms Left leg organisms (cfu/ml)found (cfu/ml) found pre-treatment 1 1.9 × 10⁸ Coliforms 4.5 × 10⁶Coliforms Proteus spp Proteus spp skin flora skin flora post-treatment 11.2 × 10⁵ Coliforms 4.5 × 10⁴ skin flora skin flora β-haemolytic Proteusspp streptococci Coliforms Proteus ssp pre-treatment 2 3.0 × 10³Coliforms 1.5 × 10⁴ coliforms Proteus spp skin flora skin florapost-treatment 2   1 × 10³ Coliforms <10 no growth Proteus spp skinflora pre-treatment 3 <10 no growth 6.3 × 10³ Coliforms β-haemolyticstreptococci skin flora post-treatment 3 3.0 × 10² Coliforms 3.0 × 10³β-haemolytic skin flora streptococci Proteus spp post 24 hours 2.7 × 10²Coliforms 1.5 × 10⁶ β-haemolytic Proteus spp streptococci skin floraColiforms Proteus spp

CONCLUSION

[0105] The main objectives of the clinical study were to examine patientcomfort and safety, as well as the efficiency of a treatment ofsuper-oxidized water based on hypochlorous acid.

[0106] The use of antiseptics for cleansing wounds is controversial,particularly with reference to the degree of pain associated with thiskind of treatment. This patient did not experience pain and, in fact,commented on a soothing effect. There was a positive effect on thebacterial flora as well as the clinical appearance of the wounds. Therewas no adverse effect on the surrounding skin which, in a number ofpatients with long-standing ulcers, is often sensitive.

[0107] While the invention has been described with reference to theexamples in relation to the treatment of leg ulcers, it should beappreciated that the invention has considerably wider applicability. Forexample, the invention has applicability to burns, to organ transplantsin relation to which current practice is to disinfect organs withantibiotics for two weeks before they are used in a patient, todisinfection of valve-replacements, and to surface wounds, open woundsand plural cavity infections which are exhibiting drug-resistance.

[0108] It will be appreciated by those skilled in the art that changescould be made to the embodiments described above without departing fromthe broad inventive concept thereof. It is understood, therefore, thatthis invention is not limited to the particular embodiments disclosed,but it is intended to cover modifications within the spirit and scope ofthe present invention as defined by the appended claims.

I claim:
 1. A method for treating an open wound in a human or animalbody comprising administering to the open wound a super-oxidized waterhaving a pH of 4 to 7 based on hypochlorous acid, wherein thesuper-oxidized water is administered in an amount effective to act as abiocide and permit cell proliferation for wound healing.
 2. The methodaccording to claim 1, wherein the open wound is an ulcer, burn, orsurface wound.
 3. The method according to claim 1, wherein thesuper-oxidized water is in a liquid form and is applied to the wound bybathing.
 4. The method according to claim 3, wherein the super-oxidizedwater is applied to the wound by immersing in a hydrobath containing thewater.
 5. The method according to claim 1, wherein the super-oxidizedwater is in a liquid form and is applied to the wound by spraying. 6.The method according to claim 1, wherein the super-oxidized water is ina gel form and is topically applied to the wound.
 7. The methodaccording to claim 1, wherein the super-oxidized water has a pH of 4.0to 6.5.
 8. The method according to claim 7, wherein the super-oxidizedwater has a pH of 4.0 to 6.2.
 9. The method according to claim 8,wherein the super-oxidized water has a pH of 4.3 to 6.2.
 10. The methodaccording to claim 9, wherein the super-oxidized water has a pH of about5.4.
 11. The method according to claim 1, wherein the super-oxidizedwater has a redox potential of >950 mV.
 12. The method according toclaim 11, wherein the super-oxidized water has a redox potential ofabout 1000 mV.
 13. The method according to claim 1, wherein thesuper-oxidized water or a preparation derived therefrom has a biociderate (D Value) of approximately 1 log reduction unit of bacillussubtilis spores in less than 1 minute with a 9:1 super-oxidized water:innoculum mix.
 14. The method according to claim 1, wherein thesuper-oxidized water is diluted to an extent that it does not inhibitcell proliferation in use.
 15. The method according to claim 14, whereinthe super-oxidized water is diluted to an extent that it promotes cellproliferation in use.
 16. The method according to claim 1, wherein thesuper-oxidized water comprises an output solution obtained byelectrochemical treatment of a saline solution.
 17. The method accordingto claim 16, wherein the super-oxidized water has a pH adjusted to adesired level by using an alkaline solution output from anelectrochemical cell in which the saline solution is treated.
 18. Themethod according to claim 16, wherein the output solution furthercomprises a phosphate buffer to adjust a pH of the solution to a desiredlevel.